This continuing project investigates the C2 and pleckstrin homology (PH) motifs, which are conserved signaling domains used as targeting modules in over 300 and 800 human proteins, respectively. Both of these motifs are activated by a second messenger then dock to a specific intracellular membrane, thereby targeting their parent proteins to the membrane surface where substrates or effectors are located. Such targeting is essential for the activation of a wide array of signaling pathways, including phosphorylation cascades, G protein circuits, calcium signaling, vesicle trafficking, phago-, endo-, and exo-cytosis, synaptic vesicle fusion and neurotransmitter release, generation of lipid-derived second messengers, control of cell growth and chemotaxis. C2 and PH domains are also linked to a wide array of human diseases ranging from cancer (C2 domain of PTEN; PH domain of protein kinase B / Akt) to inflammation (C2 domain of cytosolic phospholipase A2). Moreover, multiple C2 and PH domains are essential components of the leukocyte chemotaxis pathway responsible for tracking down invading or damaged cells marked for destruction, a pathway that is central to effective immune response. The Specific Aims of the project are to elucidate the molecular mechanisms used by C2 and PH domains to recognize and dock to their specific target membranes, to generate a structural picture of the membrane-docked states of both domains, and to develop a spatiotemporal map of the complex web of targeting events triggered by C2 and PH domains during leukocyte chemotaxis. The Progress Report describes published and preliminary results revealing the mechanisms of C2 domain calcium selectivity and activation, the mechanism of PH domain searching for its rare target lipid, and the docking geometry of membrane-bound C2 domains. Overall, the results of the proposed studies will have significant implications for a molecular understanding of C2 and PH domain signaling in human health and disease.